So similar, yet so different: the paradigm of PARP9 macro domain paralogs

Abstract

Human PARP9 harbours two tandem macro domains, MD1 and MD2, with distinct roles in ADP-ribosylation signaling. Whereas MD1 is a MacroD-type de-MARylase ("eraser"), MD2 functions as a MacroH2A-like "reader" lacking detectable hydrolase activity. How two domains so similar in sequence and fold achieve such divergent functionality has remained unclear. Our de-MARylation assays confirmed this division of labor, even though crystal structures revealed nearly identical /{beta}/ folds and similar binding pockets, and solution NMR shows broadly comparable dynamics. A remarkable distinction, however, emerges from isothermal titration calorimetry, showing that both domains bind free ADP-ribose with comparable affinity (KD = 5.4 and 8.4 M), but through markedly different thermodynamics: MD1 binding is enthalpy-driven and offset by a larger entropic penalty, whereas MD2 binds with weaker enthalpy and a smaller entropic cost. Our ADPr-bound crystal structures rationalized this showing that the distal ribose is positioned differently in the two pockets, connecting to a glycine-rich catalytic loop and a conserved aromatic residue present in active macro domains like MD1, but altered in MD2, while the catalytic asparagine itself is structurally conserved (Asn140/Asn339). Moreover, we show that a single amino acid substitution in MD2 leads to detectable RNA de-MARylation activity without altering its fold. Together, our findings show that an eraser-versus-reader distinction between the macrodomains in PARP9 is encoded in the dynamics of ligand binding and the exact position the distal ribose, revealing an unexpected catalytic plasticity relevant to PARP9s roles in immunity and cancer.